PROJECT SUMMARY NOD1 and NOD2 are pattern recognition receptors that sense fragments of bacterial peptidoglycans, and are able to detect perturbations in cellular processes such as the modulation of the actin cytoskeleton and disturbance in endoplasmic reticulum (ER) homeostasis. Under different stressful conditions, such as bacterial and viral infections, protein misfolding and perturbations in calcium homeostasis, the ER is unable to maintain homeostasis and activates the unfolded protein response (UPR). Upon ER stress three transmembrane receptors, IRE1?, PERK and ATF6 are activated and regulate biological processes such as inhibition of protein translation, autophagy, and inflammation to reestablish cellular homeostasis. NOD1 and NOD2 have been implicated in ER stress-induced inflammation, by acting downstream in the UPR to induce NF-?B activation and IL-6 production. The exact mechanism how NOD1 and NOD2 can sense ER stress is currently unknown. NOD1 and NOD2 can also sense the activation of small Rho GTPases such as Rac1. Rac1 activation leads to membrane ruffling as well as activation of the transcription factor NF-?B. We and others have shown that NOD1 and NOD2 interact with Rac1 at the cell membrane. The underlying mechanism of Rac1-mediated NOD1 and NOD2 activation is currently unknown. In the application we propose to study the mechanisms of peptidoglycan-independent activation of NOD1 and NOD2 by modulation of the actin cytoskeleton and thapsigargin-induced ER stress. Our central hypothesis is that NOD1 and NOD2 can detect cellular perturbations independent of peptidoglycan recognition. We will test key aspects of our hypothesis using the logical and innovative approach outlined in the following specific aims. Specific Aim 1. Determine the role of NOD1 and NOD2 in sensing ER stress. We will determine the contribution of calcium flux from the ER to the mitochondria in ER stress induced NOD1 and NOD2 activation. We will test our hypothesis that mitochondria damaged by ER stress release damage-associated molecular patterns (DAMPs) that activate NOD1 and NOD2. Specific Aim 2. Perturbations in cellular processes determines NOD1 and NOD2 localization. We will investigate the cellular localization of NOD1 and NOD2 in cells treated with thapsigargin to induce ER stress and in cells that express active Rac1 or Rac1 mutant forms that either induce cytoskeletal remodeling or NF-?B activation. Characterizing and understanding the mechanisms of peptidoglycan-independent NOD1 and NOD2 activation provides a plausible explanation for the observation that viruses and parasites trigger NOD1 and NOD2 signaling. These findings are innovative new concepts and would markedly influence the current concepts of NOD1 and NOD2 biology.